Abstract

Intracellular Ca²⁺ regulates numerous proteins and cellular functions and can vary substantially over submicron and submillisecond scales, so precisely localized fast detection is desirable. We have created a 1-kDa biarsenical Ca²⁺ indicator, called Calcium Green FlAsH (CaGF, 1), to probe [Ca²⁺] surrounding genetically targeted proteins. CaGF attached to a tetracysteine motif becomes ten-fold more fluorescent upon binding Ca²⁺, with a K_d of 100 M, <1-ms kinetics and good Mg²⁺ rejection. In HeLa cells expressing tetracysteine-tagged connexin 43, CaGF labels gap junctions and reports Ca²⁺ waves after injury. Total internal reflection microscopy of tetracysteine-tagged, CaGF-labeled _1C L-type calcium channels shows fast-rising depolarization-evoked Ca²⁺ transients, whose lateral nonuniformity suggests that the probability of channel opening varies greatly over micron dimensions. With moderate Ca²⁺ buffering, these transients decay surprisingly slowly, probably because most of the CaGF signal comes from closed channels feeling Ca²⁺ from a tiny minority of clustered open channels. With high Ca²⁺ buffering, CaGF signals decay as rapidly as the calcium currents, as expected for submicron Ca²⁺ domains immediately surrounding active channels. Thus CaGF can report highly localized, rapid [Ca²⁺] dynamics.

1. Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0647, USA
2. Department of Pharmacology, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0647, USA
3. Center for Theoretical Biological Physics, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0647, USA.
4. Computational Neurobiology Lab, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, California 92037, USA.
5. Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0647, USA.
6. Department of Molecular and Cellular Physiology, Stanford University School of Medicine, 279 Campus Drive, Stanford, California 94305, USA.
7. Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0647, USA.
8. Present address: HHMI Janelia Farm Research Campus, 19700 Helix Drive, Ashburn, Virginia 20147, USA.

Correspondence to: Roger Y Tsien^1,2,7 e-mail: rtsien@ucsd.edu

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